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Establishing a sensitive fluorescence-based quantification method for cyclic nucleotides.

Nadine Gruteser1, Viktoria Kohlhas1,2, Sabine Balfanz1

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This study presents a cell-free assay using purified Epac1-camps biosensor for quantifying cyclic adenosine 3

Keywords:
Cell-based assayCyclic nucleotide quantificationEpac1-campsOptogenetic sensorSignaling

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Pharmacology

Background:

  • G-protein coupled receptors (GPCRs) are drug targets for ~40% of prescribed medications.
  • GPCR activation modulates intracellular second messenger levels, such as cyclic adenosine 3',5'-monophosphate (cAMP).
  • Genetically encoded biosensors, like Epac1-camps, enable monitoring of cAMP dynamics in real-time.

Purpose of the Study:

  • To develop a cell-free assay for quantifying cAMP concentrations.
  • To evaluate the utility of purified Epac1-camps biosensor for cAMP detection.

Main Methods:

  • Bacterial over-expression and purification of Epac1-camps biosensor.
  • Development of a cell-free, multi-well plate assay for cAMP detection.
  • Assessment of biosensor sensitivity, stability, and tolerance to varying conditions.

Main Results:

  • The Epac1-camps biosensor detected as little as 0.15 pmol of cAMP.
  • Assay sensitivity remained unaffected by non-physiological salt concentrations or pH.
  • The purified Epac1-camps protein demonstrated stability during desiccation and storage.

Conclusions:

  • Purified Epac1-camps provides a robust, fast, and sensitive method for cAMP determination.
  • This cell-free assay is suitable for routine and high-throughput analysis of cAMP levels in cell lysates and tissue samples.